CN1151262C - Method for expression of foreign genes and vectors therefor - Google Patents
Method for expression of foreign genes and vectors therefor Download PDFInfo
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Abstract
A method for the expression of foreign genes enabling the stronger expression of the foreign genes than those achieved by the conventional methods. This method comprises inserting a foreign gene into the downstream of a promoter and expressing the gene in the cell, wherein two or more intron sequences which are the same or different and capable of promoting the expression of the foreign gene have been inserted into the upstream of the foreign gene.
Description
Technical field
The present invention relates to expression of heterologous genes method and used recombinant vectors thereof.More particularly, the present invention relates to the method for genetic engineering technique expression of heterologous genes in cell and relate to its used carrier, compare with existing allogeneic gene expression method, method of the present invention more can promote allogeneic gene expression.
Background technology
The promotion of allogeneic gene expression be in genetically engineered, one of necessary technology when engineered technology is used for plant particularly.
As one of these class methods, knownly will insert the method for heterologous gene upstream from the dna fragmentation of intron (intron), for example open described in the flat 3-103182 communique the spy, by inserting the heterologous gene upstream from the dna fragmentation of the intron of castor-oil plant catalase gene (CAT-1), and make this allogeneic gene expression, and promote this expression of heterologous genes.Reported the same phenomenon of relevant dna fragmentation from various introns.
In order to promote expression of heterologous genes, use intron always, but generally need not a plurality of introns, so do not see its effect yet.For example: first intron of the alcoholdehydrogenase of corn and the 6th intron manifest the effect that promotes genetic expression respectively individually, but both in conjunction with the time effect no more than the effect (Mascarenhas etc. of independent use the 6th intron, Plant Mol.Biol., 15,913-920) (1990)).Again, two maize actin the 3rd introns in conjunction with the time, its effect is also not as the effect used separately (people Mol.Gen.Genet. such as Luehrsen, 225,81-93 (1991)).
Insertion is effectively from the existing method of the dna fragmentation of intron, but it promotes that the effect of expressing is scarcely abundant, and people's expectation has the method for expressing more strongly.
Summary of the invention
Therefore, the purpose of this invention is to provide a kind of than existing method the allogeneic gene expression method of expression of heterologous genes and used for this reason recombinant vectors more consumingly.
Through making great efforts the result of research, discoveries such as the inventor, when inserting the heterologous gene upstream, will have the method that the identical or different a plurality of dna fragmentations from intron that promote the allogeneic gene expression effect insert the upstream of heterologous gene and can improve expression of heterologous genes more significantly than inserting an existing method from the dna fragmentation of intron.
Just, the invention provides a kind of method of expression of heterologous genes, comprise heterologous gene is inserted the promotor downstream and expression of heterologous genes in cell, it is characterized in that having a upstream that the dna fragmentations from intron effect, identical or inequality that promote allogeneic gene expression insert this heterologous gene, to express this heterologous gene with a plurality of.
Again, the present invention also provides recombinant vectors, and this carrier contains: promotor; The heterologous gene that inserts in its downstream; And a plurality of dna fragmentations identical or inequality that insert in the upstream of this heterologous gene, have the effect that promotes allogeneic gene expression from intron.
Discoveries such as the applicant: when the intron sequences of the corn ubiquitin of base sequence shown in the sequence number 3 in having sequence table (ュ PVC キ チ Application) is inserted into the upstream of heterologous gene individually, also have the effect that promotes this allogeneic gene expression, thereby finish second purpose of the application.
Just, the present invention also provides the method for the expression of heterologous genes with following feature, comprise heterologous gene is inserted the promotor downstream, and in cell, express this heterologous gene, it is characterized in that upstream that sequence shown in the sequence number in the sequence table 3 or its functional varient are inserted this heterologous gene, to express above-mentioned heterologous gene.
Can provide with the method for existing expression of heterologous genes by the present invention and to compare, have the method for obvious promotion expression of heterologous genes and be its used recombinant vectors.According to the present invention, can promote that therefore, in the genetically engineered field, the present invention will make big contribution with the expression of heterologous genes of gene engineering method importing.
Implement optimal mode of the present invention
The inventive method is characterised in that, by having more than two being promoted the upstream allogeneic gene expression effect, that insert the heterologous gene that will express from the dna fragmentation of intron, to promote expression of heterologous genes.
Here, so-called " have the effect that promotes allogeneic gene expression, from the dna fragmentation of intron " is meant following dna fragmentation from intron, insert this segment and do not promoted expression of heterologous genes, make expression level reach detectable degree than inserting this segment.Known have multiple such dna fragmentation from intron, for example have: (spy opens people such as flat 3-103182 communique, Tanaka to first intron of the catalase gene of castor-oil plant (CAT-1), Nucleic Acids Res.18,6767-6770 (1990)), the UDP-glucose of corn: the intron of flavonol glycosyltransferase (people such as Callis, Genes ﹠amp; Develop.1,1183-1200 (1987)), first intron (people such as Callis, the Genes ﹠amp of the alcoholdehydrogenase-1 of corn; Develop.1,1183-1200 (1987)).The second and the 6th intron of the alcoholdehydrogenase of corn-1 (people such as Mascarenhas, Plant Mol.Biol, 15,913-920 (1990)),-1 (シ ユ ラ Application Network Application-1) first intron (people such as Vasil is agree in the Shulan of corn, Plant Physiol.91,1575-1579 (1989)), first intron of the translation elongation factor EF-1 α of Arabidopis thaliana (Arabidopsis thaliana) (people such as Curie, Nucleic Acids Res.19,1305-1310 (1991)) and first intron of the Actin muscle of rice (people such as McEloy, Plant Cell2,163-171 (1990)) etc.But, the used dna fragmentation of the present invention from intron be not limited to above-mentioned these, any dna fragmentation from intron of its downstream allogeneic gene expression that can promote can use.
Again, people such as inventor is before this by comparing the intron of finding rice PLD gene with the cDNA of Phospholipase D (PLD) gene of rice and the base sequence of genomic dna; And one of find in these introns to have the effect of obvious its downstream gene expression of promotion, therefore apply for a patent (PCT/JP96/00812).The base sequence of this intron is shown in the sequence number 1 of sequence table.In the present invention, can use this dna fragmentation shown in the sequence number 1 from intron.Also can preferably use the represented castor-oil plant catalase intron sequences of in sequence table sequence number 2 and the intron sequences of corn ubiquitin (Ubiquittin) gene shown in the sequence number 3 again.
Moreover this area the professional know, and is general in having the dna sequence dna of physiological action, adds, inserts, lacks or can also keep its physiologically active when replacing one or more Nucleotide.In the present invention, the above-mentioned known dna fragmentation with the expression that promotes its downstream gene that carries out this modification gained from the dna fragmentation or the dna fragmentation from intron shown in the sequence number 1 of intron is also contained in the what is called of the present invention " from the dna fragmentation of intron ".Just, with above-mentioned known from the identical nucleotide sequence of the dna fragmentation of intron, or the nucleotides sequence shown in the sequence number 1 lists in the sequence table, adds, lacks or replace one or more Nucleotide and can promote the dna fragmentation of the expression of its downstream gene to be also contained in " from the dna fragmentation of intron " among the present invention again.Here, the dna fragmentation from intron through modifying so preferably has more than 70% with the dna fragmentation of primary from intron, more preferably the homology more than 90%.Equally, at " the functional varient " described in the application's claim also is to add on original sequence, lack or replace one or more Nucleotide and can promote the sequence of its downstream gene expression again, preferred this sequence and original series have more than 70%, and the homology more than 90% is more preferably arranged.For example: so-called " the functional varient of sequence shown in the sequence number 1 " is exactly: add, lack or replace one or more Nucleotide shown in the sequence number 1 in the sequence, can promote the sequence of its downstream gene expression, it preferably with original series, have more than 70%, the homology more than 90% more preferably arranged.
Above-mentioned each all be known from dna fragmentation of intron because of its base sequence and source, therefore, the PCR method by ordinary method just can easily prepare.Again, but length is no more than the also chemosynthesis of dna fragmentation of 2000bp.Again, the dna fragmentation from intron of above-mentioned modification also can be used site-specific mutagenesis method or chemical synthesis and preparation easily.
In the methods of the invention, a plurality of above-mentioned dna fragmentations from intron are inserted the upstream of the heterologous gene that will express, just insert the transcriptional domain of this heterologous gene, preferably insert 5 ' end of this transcriptional domain.Dna fragmentation from intron preferably inserts between promotor and the heterologous gene.Also can insert the upstream of the heterologous gene that the next-door neighbour will express from the dna fragmentation of intron; From also getting involved other sequence between the dna fragmentation of intron and the heterologous gene.The length of this intervening sequence is also unrestricted, is about 0～1000bp usually.Again, promotor directly is connected also passable with dna fragmentation from intron; There have other sequence to get involved between them to be also passable.The length of these intervening sequences is not restricted especially, is about 0～1000bp usually.
In the methods of the invention, with a plurality of, preferred 2～5, more preferably 2 or 3 above-mentioned dna fragmentations from intron insert.The dna fragmentation from intron that inserts can be an identical sequence, also can be sequence inequality.The a plurality of dna fragmentations from intron that insert also can directly connect mutually, also can get involved other sequence between them.The length of this intervening sequence is not restricted especially, is about 0～1000bp usually.
Moreover promotor also available energy makes any promotor of the allogeneic gene expression in its downstream, and preferred examples can be a 35S promoter, but also is not limited thereto.
The present invention also provides the recombinant vectors that is applicable to the invention described above method.Just, following recombinant vectors provided by the present invention, it comprises, promotor; The heterologous gene that will express that connects in its downstream; Be inserted in a plurality of dna fragmentations identical or inequality this heterologous gene upstream, that have promotion allogeneic gene expression effect from intron.Such recombinant vectors can be by inserting above-mentioned a plurality of dna fragmentation and heterologous genes from intron in the suitable expression and obtain.Because the base sequence at the clone position of expression vector is known, so by using suitable restriction enzyme and using joint can easily carry out this insertion operation in case of necessity.
Moreover various such expression vectors are well known in the art, and have commercially available.These expression vectors contain at least: the replication origin that is used for duplicating in host cell; Promotor; Selective markers such as the clone position of inserting the required restriction enzyme position of heterologous gene and drug resistance gene are provided.They contain usually makes the terminator and the SD sequence when the host is bacterium of duplicating stable termination.In the methods of the invention, can adopt any in these known expression vectors.
Discoveries such as the present application people when intron sequences base sequence, the corn ubiquitin gene is inserted in the upstream of heterologous gene individually shown in the sequence number 3 that will have sequence table, also have the effect that promotes this allogeneic gene expression.Therefore, replace above-mentioned a plurality of sequence, and situation about only the functional varient of sequence shown in the sequence number 3 or its being inserted is contained in the scope of the invention also from intron.But even use sequence shown in the sequence 3, under the situation of inserting a plurality of these sequences, its effect is stronger, and when particularly inserting with the intron sequences with the rice PLD shown in the sequence number 1, effect is good especially.
Embodiment
Below, based on embodiment the present invention is specifically described, just, the present invention is not limited to following embodiment.
Embodiment 1
In the gene of rice PLD, with the corresponding position of the terminal non-translational region of 5 of mRNA ' on have first intron (sequence number 1, WO 95/09234) of 173bp.Studied its influence with regard to this intron to genetic expression in the vegetable cell.The primer of 0 base of Synthetic 2 (5 '-TCACCACCCGGTAAGCCCAG-3 ', 3 '-CCCCCGCGTCCATCCCGCTC-5 '), each primer contains the zone from exon of 10 bases, carries out PCR with rice genomic clone (Network ロ-Application) as template.Use each 50pmol of primer, the dATP of 200 μ M, dCTP, dGTP and dTTP, 1 * PCR damping fluid (precious wine is made corporate system) and 2.5UAmpliTaq archaeal dna polymerase (precious wine is made corporate system) are to carry out the PCR reaction among the 50 μ l at cumulative volume.Reaction is carried out 30 circulations repeatedly by following temperature condition, that is, in DNA thermal cycler (PARKIN ELMER CETUS corporate system), 94 ℃ one minute, 40 ℃ one minute and be two minutes and 30 seconds at 72 ℃.
With PCR product subclone to PCR II carrier (INVITROGEN corporate system), the ECoRI fragment that therefrom cuts out is carried out after smooth end handles, insert plasmid pBI221[that Japan spins company's preparation inserts beta-Glucuronidase gene (hereinafter referred to as " GUS ") in the downstream of 35S promoter (hereinafter referred to as " 35Spro ") vector plasmid] the SmaI site on, obtain carrier pBI221P (35S pro, the PLD intron, GUS).Again this plasmid is digested with BamHI, the gained segment is carried out above-mentioned fragment being embedded after smooth end handles, and preparation carrier pBI221PP (35S pro, PLD intron * 2, GUS).
With Xbal the spy being opened the above pIG221[of flat 03-103182 communique again has castor-oil plant catalase intron (sequence number 2 in the sequence table) and GUS to exist in this order in the downstream of 35S pro] digest, the gained fragment is carried out after smooth end handles, the dna fragmentation that will have above-mentioned PLD intron sequences inserts, make carrier pIG221P (35S pro, the PLD intron, the catalase intron, GUS).
Immature embryo system by Japanese rice " Japanese Ba Lei " kind becomes rice culturing cell (people such as Hiei, The Plant Journal, 6,271-282 (1994)], according to existing report [island people such as this grade, Nature, 338,274-276 (1989)] in these cells, after the importing said gene, measure beta-Glucuronidase (GUS) activity, the results are shown in table 1.
As shown in table 1 such, as can be seen: compare with using single intron, import two kinds of identical or different introns, the activity of the GUS of obvious increase is arranged.Simultaneously, clearly, the dna fragmentation of base sequence that the PLD intron is arranged can obtain to promote the effect of genetic expression when using the dna fragmentation of a plurality of base sequences with intron.
Table 1
| Plasmid | The GUS activity |
| No pBI221 (35S pro, GUS) pBI221P (35S pro, the PLD intron, GUS) pBI22PP (35S pro, PLD intron * 2, GUS) pIG221 (35S pro, the catalase intron, GUS) pIG221P (35S pro, the PLD intron, the catalase intron, GUS) | 7.2 14 180 430 160 680 |
Embodiment 2
With regard to the intron (people such as Ubi-1:Christensen A.H., Plant Mol.Biol., 18,675-689 (1982)) of the ubiquitin gene of corn, study its facilitation effect to allogeneic gene expression, used carrier has two kinds.
The carrier of effect when at first, being ready for use on the single intron of research insertion in order to the below legal system.Cut out the intron (sequence number 3) of promotor and ubiquitin gene with PstI, insert the PstI site of pUC18 carrier.Cut out the intron part with BglII and BamHI, be inserted into the BamHI site of pBI221 carrier, make carrier pBI221U (35S pro, ubiquitin intron, GUS).
The carrier of effect when secondly, being ready for use on a plurality of intron of research insertion in order to the below legal system.Cut out the intron part with BglII and BamHI, after smooth end is handled, be inserted into the SmaI site of pBI221 carrier.Carry out smooth end again in the BamHI site of this carrier and handle, described PLD intron is inserted, thus make carrier pBI221PU (35S pro, the PLD intron, the ubiquitin intron, GUS).With aforesaid method these carriers are imported bioblast, measure the GUS activity.
Just as shown in table 2, can find out and use single ubiquitin intron that the effect that promotes that GUS expresses is arranged.Can find out and with PLD intron and ubiquitin intron, than using single intron to have stronger facilitation effect again.
Table 2
| Plasmid | GUS activity (pmol MU/min./mg protein) |
| No pBI221 (35S pro, GUS) pBI221 (35S pro, the PLD intron, GUS) pBI221 (35S pro, ubiquitin intron, GUS) pBI221 (35S pro, PLD intron, the ubiquitin intron, GUS) | 3.3 |
| 13 | |
| 100 | |
| 360 | |
| 780 |
Below be sequence table:
Sequence table
Sequence number: 1
Sequence length: 173
Sequence type: nucleic acid
Sequence kind: genomic DNA
The source
Biological name: rice (Oryza Sativa)
Topology: straight chain shape
Chain number: two strands
Sequence
GTAAGCCCAG TGTGCTTAGG CTAAGCGCAC TAGAGCTTCT TGCTCGCTTG CTTCTTCTCC 60
GCTCAGATCT GCTTGCTTGC TTGCTTCGCT AGAACCCTAC TCTGTGCTGC GAGTGTCGCT 120
GCTTCGTCTT CCTTCCTCAA GTTCGATCTG ATTGTGTGTG TGGGGGGGCG CAG 173
Sequence number: 2
Sequence length: 217
Sequence type: nucleic acid
Sequence kind: genomic DNA
The source
Biological name: castor-oil plant
Topology: straight chain shape
Chain number: two strands
Sequence
ACATGGATCC CTACAGGGTA AATTTCTAGT TTTTCTCCTT CATTTTCTTG GTTAGGACCC 60
TTTTCTCTTT TTATTTTTTT GAGCTTTGAT CTTTCTTTAA ACTGATCTAT TTTTTAATTG 120
ATTGGTTATG GTGTAAATAT TACATAGCTT TAACTGATAA TCTGATTACT TTATTTCGTG 180
TGTCTATGAT GATGATGATA GTTACAGAAC CGTCGAC 217
Sequence number: 3
Sequence length: 1010
Sequence type: nucleic acid
Sequence kind: genomic DNA
The source
Biological name: corn
Topology: straight chain shape
Chain number: two strands
Sequence:
GTACGCCGCT CGTCCTCCCC CCCCCCCCCT CTCTACCTTC TCTAGATCGG CGTTCCGGTC 60
CATGGTTAGG GCCCGGTAGT TCTACTTCTG TTCATGTTTG TGTTAGATCC GTGTTTGTGT 120
TAGATCCGTG CTGCTAGCGT TCGTACACGG ATGCGACCTG TACGTCAGAC ACGTTCTGAT 180
TGCTAACTTG CCAGTGTTTC TCTTTGGGGA ATCCTGGGAT GGCTCTAGCC GTTCCGCAGA 240
CGGGATCGAT TTCATGATTT TTTTTGTTTC GTTGCATAGG GTTTGGTTTG CCCTTTTCCT 300
TTATTTCAAT ATATGCCGTG CACTTGTTTG TCGGGTCATC TTTTCATGCT TTTTTTTGTC 360
TTGGTTGTGA TGATGTGGTC TGGTTGGGCG GTCGTTCTAG ATCGGAGTAG AATTCTGTTT 420
CAAACTACCT GGTGGATTTA TTAATTTTGG ATCTGTATGT GTGTGCCATA CATATTCATA 480
GTTACGAATT GAAGATGATG GATGGAAATA TCGATCTAGG ATAGGTATAC ATGTTGATGC 540
GGGTTTTACT GATGCATATA CAGAGATGCT TTTTGTTCGC TTGGTTGTGA TGATGTGGTG 600
TGGTTGGGCG GTCGTTCATT CGTTCTAGAT CGGAGTAGAA TACTGTTTCA AACTACCTGG 660
TGTATTTATT AATTTTGGAA CTGTATGTGT GTGTCATACA TCTTCATAGT TACGAGTTTA 720
AGATGGATGG AAATATCGAT CTAGGATAGG TATACATGTT GATGTGGGTT TTACTGATGC 780
ATATACATGA TGGCATATGC AGCATCTATT CATATGCTCT AACCTTGAGT ACCTATCTAT 840
TATAATAAAC AAGTATGTTT TATAATTATT TTGATCTTGA TATACTTGGA TGATGGCATA 900
TGCAGCAGCT ATATGTGGAT TTTTTTAGCC CTGCCTTCAT ACGCTATTTA TTTGCTTGGT 960
ACTGTTTCTT TTGTCGATGC TCACCCTGTT GTTTGGTGTT ACTTCTGCAG 1010
Claims (26)
1. the method for expression of heterologous genes, comprise the downstream of heterologous gene being inserted promotor, and this heterologous gene is expressed in cell, it is characterized in that, 2-5 can be promoted allogeneic gene expression, the upstream of inserting this heterologous gene from the dna fragmentation identical or inequality of intron, to express described heterologous gene, described dna fragmentation from intron has at least one to comprise the sequence that is selected from down group: sequence shown in the sequence number 1 or its functional varient, sequence shown in the sequence number 2 or its functional varient, sequence shown in the sequence number 3 or its functional varient, and, in sequence shown in the sequence number 1, sequence shown in the sequence number 2, sequence shown in the sequence number 3 is added in any, lack or replace the resulting sequence of one or more Nucleotide or its functional varient.
2. the described method of claim 1, wherein the dna fragmentation from intron is 2 or 3.
3. claim 1 or 2 described methods, wherein said dna fragmentation from intron has at least one to comprise the sequence that is selected from down group: sequence shown in the sequence number 1 or its functional varient, sequence shown in the sequence number 2 or its functional varient, sequence shown in the sequence number 3 or its functional varient, and, add, lack or replace the resulting sequence of one or more Nucleotide or its functional varient in any in sequence shown in sequence, the sequence number 3 shown in sequence shown in the sequence number 1, the sequence number 2.
4. claim 1 or 2 described methods is characterized in that, described dna fragmentation from intron is to insert between above-mentioned promotor and above-mentioned heterologous gene.
5. each described method in the claim 1 to 4 is characterized in that described cell is a vegetable cell.
6. any one described method of claim 1 to 5 is characterized in that, in described dna fragmentation from intron, has at least one to contain the sequence shown in the sequence number 1 or its functional varient in the ordered list.
7. the described method of claim 6 is characterized in that, described dna fragmentation from intron contains 2 sequence or its functional varients shown in sequence number in the sequence table 1.
8. any one described method in the claim 1 to 6 is characterized in that, in described dna fragmentation from intron, at least one contains sequence shown in the sequence number 2 or its functional varient in the ordered list.
9. the method put down in writing of claim 8 is characterized in that described dna fragmentation from intron contains sequence or its functional varient shown in sequence shown in the sequence number 1 or its functional varient and the sequence number 2.
10. any one described method in the claim 1 to 5 is characterized in that, in the described dna fragmentation from intron, has at least one to contain sequence shown in the sequence number 3 or its functional varient in the ordered list.
11. the described method of claim 10 is characterized in that, described dna fragmentation from intron contains sequence or its functional varient shown in sequence shown in the sequence number 1 or its functional varient and the sequence number 3.
12. recombinant vectors, it is characterized in that, contain promotor, with the heterologous gene that will express that inserts this promotor downstream, and insert this heterologous gene upstream, can promote allogeneic gene expression, 2-5, identical or inequality, dna fragmentation from intron, described dna fragmentation from intron has at least one to comprise the sequence that is selected from down group: sequence shown in the sequence number 1 or its functional varient, sequence shown in the sequence number 2 or its functional varient, sequence shown in the sequence number 3 or its functional varient, and, in sequence shown in the sequence number 1, sequence shown in the sequence number 2, sequence shown in the sequence number 3 is added in any, lack or replace the resulting sequence of one or more Nucleotide or its functional varient.
13. the described recombinant vectors of claim 12, wherein the dna fragmentation from intron is 2 or 3.
14. claim 12 or 13 described recombinant vectorss, wherein said dna fragmentation from intron has at least one to comprise the sequence that is selected from down group: sequence shown in the sequence number 1 or its functional varient, sequence shown in the sequence number 2 or its functional varient, sequence shown in the sequence number 3 or its functional varient, and, add, lack or replace the resulting sequence of one or more Nucleotide or its functional varient in any in sequence shown in sequence, the sequence number 3 shown in sequence shown in the sequence number 1, the sequence number 2.
15. claim 12 or 14 described recombinant vectorss is characterized in that, described dna fragmentation from intron is to be inserted between described promotor and the described heterologous gene.
16. each described recombinant vectors is characterized in that among the claim 12-15, this recombinant vectors can duplicate in vegetable cell.
17. any one described recombinant vectors is characterized in that in the claim 12 to 16, in the described dna fragmentation from intron, has at least one to contain the sequence shown in the sequence number 1 or its functional varient in the ordered list.
18. the described recombinant vectors of claim 17 is characterized in that, described dna fragmentation from intron contains 2 sequence or its functional varients shown in sequence number in the sequence table 1.
19. any one described recombinant vectors is characterized in that in the claim 12 to 16, in the described dna fragmentation from intron, has at least one to contain the sequence shown in the sequence number 2 or its functional varient in the ordered list.
20. the described recombinant vectors of claim 19 is characterized in that, described dna fragmentation from intron contains sequence or its functional varient shown in sequence shown in the sequence number 1 or its functional varient and the sequence number 2.
21. any one described recombinant vectors is characterized in that in the claim 12 to 16, in the described dna fragmentation from intron, has at least one to contain the sequence shown in the sequence number 3 or its functional varient in the ordered list.
22. the described recombinant vectors of claim 21 is characterized in that, described dna fragmentation from intron contains sequence or its functional varient shown in sequence shown in the sequence number 1 or its functional varient and the sequence number 3.
23. the method for expression of heterologous genes, comprise heterologous gene is inserted the downstream of promotor and this heterologous gene is expressed in cell, it is characterized in that the upstream that sequence shown in the sequence number in the sequence table 3 or its functional varient are inserted this heterologous gene is to express described heterologous gene.
24. the described method of claim 23 is characterized in that, insertion be the sequence shown in the sequence number 3 in the sequence table.
25. claim 23 or 24 described methods is characterized in that, sequence shown in the sequence number in the sequence table 3 or its functional varient are inserted between described promotor and the described heterologous gene.
26. any one described method is characterized in that in the claim 23 to 25, described cell is a vegetable cell.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP172922/96 | 1996-06-12 | ||
| JP17292296 | 1996-06-12 | ||
| JP172922/1996 | 1996-06-12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1195376A CN1195376A (en) | 1998-10-07 |
| CN1151262C true CN1151262C (en) | 2004-05-26 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB971906874A Expired - Fee Related CN1151262C (en) | 1996-06-12 | 1997-06-12 | Method for expression of foreign genes and vectors therefor |
Country Status (12)
| Country | Link |
|---|---|
| US (1) | US6214578B1 (en) |
| EP (2) | EP1350850A3 (en) |
| JP (1) | JP3413207B2 (en) |
| KR (1) | KR100517818B1 (en) |
| CN (1) | CN1151262C (en) |
| AT (1) | ATE319841T1 (en) |
| AU (1) | AU732422B2 (en) |
| CA (1) | CA2226945A1 (en) |
| DE (1) | DE69735420T2 (en) |
| DK (1) | DK0846770T3 (en) |
| ES (1) | ES2259191T3 (en) |
| WO (1) | WO1997047755A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0953647B1 (en) * | 1996-12-16 | 2008-05-28 | Eisai R&D Management Co., Ltd. | Method for preparing retrovirus vector for gene therapy |
| JP2000152785A (en) * | 1998-11-19 | 2000-06-06 | Japan Tobacco Inc | Nucleic acid fragment, recombinant vector containing the same, and promotion of expression of structural gene using the same |
| SE9901339D0 (en) * | 1999-04-15 | 1999-04-15 | Astra Ab | New expression systems |
| AU784292B2 (en) * | 1999-09-27 | 2006-03-02 | Japan Tobacco Inc. | Nucleic acid fragment, recombinant vector containing the same and method of promoting the expression of structural gene by using the same |
| AU1046801A (en) * | 1999-11-05 | 2001-05-14 | South African Sugar Association | A high level, stable, constitutive promoter element for plants |
| AU2001288478B2 (en) | 2000-08-25 | 2006-11-02 | Basf Plant Science Gmbh | Plant polynucleotides encoding prenyl proteases |
| ZA200607843B (en) * | 2004-02-23 | 2008-06-25 | Israel State | Engrafted plants resistant to viral diseases and methods of producing same |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1339684C (en) | 1988-05-17 | 1998-02-24 | Peter H. Quail | Plant ubquitin promoter system |
| JPH03103182A (en) | 1989-09-14 | 1991-04-30 | Mitsubishi Kasei Corp | Dna fragment to promote manifestation of adventitious gene |
| JP3103182B2 (en) | 1992-01-22 | 2000-10-23 | ユニチカ株式会社 | Whisker-like carbon fiber |
| ES2226029T3 (en) * | 1993-06-10 | 2005-03-16 | Bayer Corporation | VECTOR AND MAMMER CELL LINE WITH IMPROVED PRODUCTIVITY. |
| EP0685559B1 (en) * | 1993-09-30 | 2004-12-01 | Japan Tobacco Inc. | Plant-derived phospholipase d gene |
| CN1174098C (en) * | 1995-03-29 | 2004-11-03 | 日本烟草产业株式会社 | DNA fragment, recombination vector containing the same, and method of the expression of alien gene with the use of the same |
| AU727951B2 (en) | 1996-02-21 | 2001-01-04 | Japan Tobacco Inc. | Method of changing the composition of the phospholipid produced by the living body and recombinant vector therefor |
-
1997
- 1997-06-12 JP JP50144698A patent/JP3413207B2/en not_active Expired - Fee Related
- 1997-06-12 DK DK97926233T patent/DK0846770T3/en active
- 1997-06-12 EP EP03015445A patent/EP1350850A3/en not_active Withdrawn
- 1997-06-12 AU AU31065/97A patent/AU732422B2/en not_active Ceased
- 1997-06-12 WO PCT/JP1997/002030 patent/WO1997047755A1/en active IP Right Grant
- 1997-06-12 DE DE69735420T patent/DE69735420T2/en not_active Expired - Fee Related
- 1997-06-12 EP EP97926233A patent/EP0846770B1/en not_active Expired - Lifetime
- 1997-06-12 KR KR19980700871A patent/KR100517818B1/en not_active IP Right Cessation
- 1997-06-12 US US09/011,526 patent/US6214578B1/en not_active Expired - Fee Related
- 1997-06-12 CN CNB971906874A patent/CN1151262C/en not_active Expired - Fee Related
- 1997-06-12 CA CA002226945A patent/CA2226945A1/en not_active Abandoned
- 1997-06-12 AT AT97926233T patent/ATE319841T1/en not_active IP Right Cessation
- 1997-06-12 ES ES97926233T patent/ES2259191T3/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| AU3106597A (en) | 1998-01-07 |
| EP1350850A3 (en) | 2003-11-19 |
| KR100517818B1 (en) | 2005-12-07 |
| DE69735420D1 (en) | 2006-05-04 |
| JP3413207B2 (en) | 2003-06-03 |
| EP0846770A1 (en) | 1998-06-10 |
| EP0846770A4 (en) | 2003-02-05 |
| CN1195376A (en) | 1998-10-07 |
| AU732422B2 (en) | 2001-04-26 |
| ATE319841T1 (en) | 2006-03-15 |
| US6214578B1 (en) | 2001-04-10 |
| DK0846770T3 (en) | 2006-07-10 |
| CA2226945A1 (en) | 1997-12-18 |
| WO1997047755A1 (en) | 1997-12-18 |
| EP0846770B1 (en) | 2006-03-08 |
| KR19990036203A (en) | 1999-05-25 |
| DE69735420T2 (en) | 2006-11-16 |
| ES2259191T3 (en) | 2006-09-16 |
| EP1350850A2 (en) | 2003-10-08 |
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